1a.Objectives (from AD-416):
The primary purpose of this agreement is to establish a general framework for cooperation and establish a mechanism by which ABADRU and USAMRIID will collaborate in research on the potential for Rift Valley Fever Virus transmission by arthropod vectors in North America. It also establishes a relationship whereby each research entity can call upon the other for advice concerning research methodology and directions.

1b.Approach (from AD-416):
A basic understanding of North American mosquito species susceptibility to Rift Valley fever virus infection and the subsequent interactions is needed in the event of an outbreak in the United States. To understand the propagation of Rift Valley fever in mosquitoes, three components will be examined in this agreement..1)The effects of temperature on virus movement within the mosquito also known as the extrinsic incubation period;.2)Whether there is reassortment of Rift Valley fever viral genes within mosquitoes; and.3)The extent of transovarial transmission or vertical transmission of Rift Valley fever virus in North American mosquitoes from infected females to their offspring.

Culex tarsalis mosquitoes were allowed to feed on a hamster with a RVF viremia. After completing engorgement, the mosquitoes were randomly aliquoted to cardboard cages that were then placed in incubators maintained at 14, 18, 22, and 26°C (a previous study conducted earlier in the year placed mosquitoes at 22, 26, and 30°C). These mosquitoes were sampled (legs and bodies separately to determine both infection and dissemination rates) at selected time periods ranging from 7-28 days. Also, a previous study indicated that virtually all Cx. tarsalis with a disseminated RVFV infection were able to transmit this virus by bite, so all Cx. tarsalis in this study with a disseminated infection were assumed to be able to transmit RVFV. Briefly, both apparent infection rates as well as viral dissemination rates were directly related to holding temperature with mosquitoes held at 14°C engorging (See Table 1).

b. Test for reassortment between two different strains of Rift Valley fever after passage through a mosquito disease vector:

Selected strains for RVFV were grown in either cell culture, or passaged in a hamster, placed in TRIzol-LS, and shipped to the USDA/ARS laboratory in Manhattan, KS, for analysis.

c. Test for vertical transmission of Rift Valley fever virus in North American mosquitoes.

Various studies have looked at the potential for mosquitoes to vertically transmit RVFV for the past 40 years. The only study that was able to demonstrate it was by Linthicum et al. and they detected RVFV in adults reared from field-collected larvae in Kenya. No laboratory study has been successful. However, the failure to demonstrate this mode of transmission is more likely due to complications in the laboratory (i.e., missing a key step that occurs in nature) than to vertical transmission not occurring. In all likelihood, vertical transmission is critical to the long-term survival of RVFV in an area, and thus it would play a crucial role in the potential for RVFV to become established in North America. Researchers tried several novel approaches (i.e., simply infecting adult mosquitoes with RVFV and collecting eggs has been tried unsuccessfully for a number of mosquitoes). These include allowing mosquito larvae to feed on tissues from a RVFV-infected hamster, pupate, feed on a naïve hamster, and lay eggs (This has previously been shown to be able to infect mosquitoes). The idea behind this is that not only does the virus need to infect the mosquito (which RVFV does exceptionally well), but more importantly, it needs to infect the germinal tissue (which it apparently does not do well – in might not be able to cross an ovarian membrane barrier). The idea behind this approach is that the tissue destined to become the ovaries becomes infected in the larval stage, before the membrane is produced, thus allowing for virus to get into the germinal tissue. Researchers have fed the larvae on liver tissue from infected hamsters, collected the adults, fed them on a naïve hamster (the hamster died indicating that some of the mosquitoes were infected), and collected eggs from these mosquitoes. Researchers will be hatching these eggs shortly to see if any of the progeny are infected. In a second approach, researchers tried inoculating Ae. albopictus and Ae. taeniorhynchus larvae directly with RVFV (this has the advantage in that researchers would know that each female mosquito derived from these larvae would be infected). Unfortunately, despite pupating normally, researchers were not able to get sufficient adult mosquitoes from these pupae to collect eggs.